Diversity through semisynthesis: the chemistry and biological activity of semisynthetic epothilone derivatives
Summary (3 min read)
Introduction
- MSA can be divided into two distinct functional classes, namely compounds that inhibit the assembly of soluble tubulin into microtubule polymers ("tubulin polymerization inhibitors") and those that promote the assembly of tubulin heterodimers into microtubule polymers and stabilize microtubules ("microtubule stabilizers") [4] .
- After the elucidation of taxol's mode of action in 1979 [6] , it took more than a decade before other microtubulestabilizing agents with non-taxol-like structures were discovered.
- Even for the natural product taxol [38] , the sustained supply of sufficient quantities of drug material for clinical use could only be secured for some time through the development of a semisynthetic production process from another natural product, namely 10-deacetylbaccatin III [39, 40] .
- This will facilitate the comparison of the biological effects of related structural changes.
Modifications of the epoxide moiety
- Modifications of the epoxide moiety have been an important trait of the semisynthetic work on epothilones from the very beginning, which is unsurprising in light of the multitude of transformations that are conceivable for an oxirane ring and the potential for further elaboration of the initial reaction products.
- In contrast to the above acetonides, cis and trans diols 9 and 5 did not show any appreciable biological activity (IC 50 's for cancer cell growth inhibition >1 µM) [42, 43] .
- As illustrated in Scheme 4, 12 was obtained through nucleophilic ring-opening of the epoxide moiety in Epo A with azide anion (to produce 11) and subsequent reduction of the azide group under Staudinger conditions (Ph 3 P/H 2 O) [48] .
- These findings re-confirmed the notion that the oxirane ring system in epothilones merely serves to stabilize the proper bioactive conformation of the macrocyclic skeleton rather than acting as a reactive electrophile or a hydrogen bond acceptor.
- Several of these derivatives show antiproliferative activities that are comparable with or even superior to that of Epo A [45] .
Modifications of the ester moiety
- One of the most obvious modifications of the epothilone scaffold is the hydrolysis of the ester bond to produce the corresponding seco acid.
- This transformation has been achieved by treatment of Epo A with NaOH/MeOH, which gave seco acid 25 in 65% yield (Scheme 8); however, ester hydrolysis was also accompanied by the retro aldol cleavage of the C3-C4 bond, thus leading to 23% of the retro aldol product 26 (Scheme 8) [58] .
- While the situation is more complex in vivo and the stability of Epo B is much higher in human than in rodent plasma, their findings on the reduced activity of epothilones in the presence of mouse plasma have led the BMS group to pursue lactambased epothilone analogs as metabolically more stable alternatives to the natural macrolactones [41, 59] .
- Using this chemistry, the BMS group has also developed an efficient one-pot process for the conversion of Epo B into lactam derivative 34, which involves the above Pd(0)-catalyzed ring-opening reaction, reduction of the azide with trimethyl phosphine and macrolactam formation with EDCI/HOBt [59] ; this provides the desired lactam in 23% overall yield.
- In spite of its limited effects against highly multidrug-resistant cell lines in vitro, intriguingly, the compound has also been found to be superior to Taxol in Taxol -resistant tumor models [62] .
Modifications in the C2-C8 region
- Semisynthesis-based modifications in the C2-C8 region have involved transformations of all functional groups present in this sector of the epothilone structure, including the hydroxyl groups at C3 and C7 as well as the keto group at C5.
- After TES protection of 37 and 38, to give 39 and 40, 1,4-addition of cyanide ion produced a ca. 1/1 mixture of 3S and 3R isomers, which were separated and then deprotected with acetic acid individually to provide 41 and 42 (as well as their corresponding 3R isomers).
- The in vitro activity of analogs 37/41 and 38/42 is less than one order of magnitude lower than that of Epo A and B, respectively; for 38 and 42 their in vitro antiproliferative activity is thus comparable with taxol [44] .
- No oxidation of the 7-OH group takes place under these conditions, but the oxidized product is obtained as its C7-methylthiomethyl (MTM) ether 44 (30% yield; Scheme 11).
Side chain modifications
- The C15 side chain of epothilones has been targeted for semisynthesis in a number of different ways that include modifications (or replacement) of the heterocycle, the vinyl linker between the heterocycle and the macrolactone ring, or both.
- Both Epo E and F have been elaborated into different C21-modified derivatives [66] and C20 substituents of limited size (but still larger than the natural methyl group) have been found compatible with potent antiproliferative activity; more bulky substituents result in a substantial loss in potency.
- ABJ879 (55) has demonstrated potent antitumor activity in experimental animal models [69] , where it produced transient regressions and inhibition of tumor growth of slowgrowing (NCI H-596 lung adenocarcinomas, HT-29 colon tumors) as well as fast-growing, difficult-to-treat tumors (NCI H-460 large cell lung tumors).
- In contrast, olefination reactions involving the C16 keto group proved to be highly problematic.
- In order to enable the replacement of the thiazole ring by other aryl moieties, Höfle and co-workers have developed an alternative strategy for the construction of the aryl-vinyl part of the epothilone side chain from ketone 59 (Scheme 17).
Replacement of the C13-O16 segment
- Semisynthetic epothilone analogs have also been prepared via intermediates that were obtained through the degradative removal of the entire C13-O16 segment (including the pendant side chain), which were then (re)elaborated into modified versions of the original structure.
- Treatment of the crude keto aldehyde thus obtained with K 2 CO 3 led to facile elimination of the C1-C12 segment as the free carboxylic acid, which was converted to ester 77 with TMS-diazomethane.
- Oxidation of 82 with NaClO 2 gave the mono-ester of a dicarboxylic acid, which could be coupled with amines 83 or 84, to give the C12-C13 amide-based analogs 85 and 86, respectively, after ester saponification, Yamaguchi-type macrolactonization and final deprotection with CF 3 COOH (Scheme 20) [41] .
- Unfortunately, none of the derivatives 85-88 showed any significant tubulin-polymerizing or growth-inhibitory activity.
Conclusions
- The work discussed in this review article has defined the chemistry associated with the epothilone molecular framework in significant detail.
- Most importantly, these efforts have led to the discovery of three derivatives that have been advanced to clinical studies in humans.
- Thus, notwithstanding the wealth of fully synthetic analogs that have been prepared over the last 15 years and many of which exhibit very attractive biological profiles (at least in vitro), semisynthesis so far has had the more profound impact on the clinical advancement of the epothilone class of microtubule stabilizers than total synthesis.
- At the same time, it is clear that the potential of semisynthesis for the creation of new structurally unique epothilone analogs is far from being exhausted.
- It is unclear at this point, whether those groups with a sufficient supply of natural epothilones (BMS, GBF (now Helmholtz Centre for Infection Research), Novartis) continue to be active in the area of epothilone semisynthesis.
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Cites background from "Diversity through semisynthesis: th..."
...For further details on the epothilones and their mechanisms of action, see the latest review by Ferrandina and colleagues [64] in addition to those by Danishefsky [45] and Altmann and colleagues [6], which describe opportunities for the synthesis of epothilones and other derivatives....
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References
128 citations
"Diversity through semisynthesis: th..." refers background or methods or result in this paper
...While the situation is more complex in vivo and the stability of Epo B is much higher in human than in rodent plasma, their findings on the reduced activity of epothilones in the presence of mouse plasma have led the BMS group to pursue lactambased epothilone analogs as metabolically more stable alternatives to the natural macrolactones [41,59]....
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...42 nM, respectively, for 34 and Epo B [59])....
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...Using this chemistry, the BMS group has also developed an efficient one-pot process for the conversion of Epo B into lactam derivative 34, which involves the above Pd(0)-catalyzed ring-opening reaction, reduction of the azide with trimethyl phosphine and macrolactam formation with EDCI/HOBt [59]; this provides the desired lactam in 23% overall yield....
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...These reports are in line with observations by the BMS group on the significantly diminished in vitro cytotoxicity of epothilones when preincubated with mouse plasma, which leads to rapid ester cleavage [59]....
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...one order of magnitude lower than that of Epo B [23,59] (e....
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127 citations
"Diversity through semisynthesis: th..." refers background or methods in this paper
...Alternatively, reaction of E-64 with N-iodosuccinimide (NIS) gave the corresponding vinyl iodide, which underwent smooth Stille coupling with stannane 67 to give 68 [71,75]....
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...While 59 could be converted to methylene derivative 60 in a modest overall yield of 15% (after deprotection), all attempts to re-introduce the natural thiazole side chain or to create a phenyl-based Epo A analog using Wittig-type chemistry were unsuccessful [47,71,75]....
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...In fact, one of the epothilone-type clinical development compounds (ZKEpo, sagopilone) is an analog of Epo B with a benzothiazole side chain (analogous to the benzoxazole side chain in analog 81) [75]; however, sagopilone could not be prepared...
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...While 64 was obtained as a 7/3 mixture of E/Z double bond isomers, these could be separated by preparative HPLC and the pure E-isomer was converted to the protected phenyl-based Epo A analog 66 through Suzuki coupling [71,75]....
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...All analogs depicted in Scheme 16 were found to be significantly less active in proliferation or tubulin assays than Epo A [47,71,75]....
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121 citations
"Diversity through semisynthesis: th..." refers background in this paper
...[56], the replacement of the epoxide moiety by a cyclopropane ring also produces enhanced binding to stabilized microtubules in vitro....
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Frequently Asked Questions (15)
Q2. How did the thiazole side chain be converted to methylene?
While 59 could be converted to methylene derivative 60 in a modest overall yield of 15% (after deprotection), all attempts to re-introduce the natural thiazole side chain or to create a phenyl-based Epo A analog using Wittig-type chemistry were unsuccessful [47,71,75].
Q3. What is the important aspect of the biochemical activity of epothilones?
The earliest contributions to this area stem from the GBF group and involved the transformation of epothilonesA, B, and C (vide infra) into a variety of C12/C13-modified derivatives [23].
Q4. What are the prominent of these new microtubule stabilizers?
Most prominent among these new microtubule stabilizers are the epothilones, which are bacteria-derived macrolides whose microtubule-stabilizing properties were discovered in 1996 by a group at Merck Research Laboratories [7]; the compounds themselves, however, had been first isolated 9 years earlier from the myxobacterium Sorangium cellulosum Sc 90 by Reichenbach and Höfle (Fig. 2) [8,9].
Q5. What is the resulting derivative of Epo A?
The resulting N-unsubstituted 12,13-aziridinyl-Epo A 21 has been converted into a series of N-substituted derivatives via alkylation, acylation, carbamoylation, or sulfonylation.
Q6. How much of the epothilone derivatives can be obtained by electrophilic?
While not accessible by base treatment and subsequent electrophilic quenching, C21-substituted epothilone derivatives can nevertheless be obtained through semisynthesis in a very efficient manner.
Q7. What is the simplest method of establishing the controlled degradation of Epo C into ester?
Employing PLE-catalyzed hydrolysis of the lactone group and subsequent cleavage of the C12/C13 double bond by ozonolysis, the BMS group was able to establish the controlled degradation of Epo C into ester 82 (Scheme 20).
Q8. What is the way to reduce the hydroxyl group in Epo A?
Selective oxidation of the hydroxyl group on C3 in Epo A is more difficult and could only be accomplished in very moderate yield with a mixture of dimethylsulfide and dibenzoylperoxide [47].
Q9. What is the IC50 value of oxazoline 13?
The phenyl-substituted oxazoline 13 was found to inhibit human cancer cell growth in vitro with IC50 values around 20 nM [48]; thus, the activity of this analog is within a 10- fold range of the activity of Epo A and it is comparable with the activity of cyclic acetals 5a and 9a (vide supra).
Q10. What is the effect of the pyridyl derivative 14 on cell growth?
It is, therefore, unclear to what extent (if at all) the enhanced cellular activity of 14 (over 13) is a result of higher affinity interactions with the tubulin/microtubule system (possibly through H-bond formation between the pyridine nitrogen and a donor group on the protein).
Q11. What is the way to convert Epo A to a whole range of analogs?
In addition to cyclopropyl-epothilones, the BMS group has also devised a strategy for the conversion of Epo A to a whole range of analogs incorporating a (substituted) aziridine ring in place of the epoxide moiety [45].
Q12. What is the IC50 of the cyclopropyl-epo B?
Bis-substitution of the oxygen-replacing carbon in cyclopropyl-Epo B by bulky bromine substituents leads to reduced activity, but the resulting analog is still more potent than Epo D against the HCT-116 cell line (IC50 against HCT116 of 3.8 nM versus 6.5 nM for Epo D [49]).
Q13. What is the first implementation of this concept?
The first implementation of this concept was reported by the GBF group, who used ring-opening olefin metathesis (ROM) with ethylene for the conversion of Epo C into the ring-opened product 69 in 73% yield (employing Grubbs II catalyst) [73] (Scheme 18).
Q14. What is the reason for the bias towards semisynthesis?
This bias towards semisynthesis reflects the technical (fewer chemical steps) and economic (cost of goods) advantages still associated with natural product derivatization.
Q15. What is the role of the epothilone in the treatment of cancer?
Epo B and a number of its analogs have been demonstrated to possess potent in vivo antitumor activity and at least seven epothilone-type compounds have entered clinical evaluation in humans (although several of these are not anymore under development).